The goal of this proposal is to understand how changes occurring during metastasis of non-small cell lung cancers (NSCLC) promote aggressive phenotypes and drug resistance. Comparison of cell line panels derived from non-metastatic versus highly metastatic tumors of the KrasLA1/+; P53R172H?G/+ (KP) mouse model, which simulates human NSCLC, identified upregulation of Musashi-2 (MSI2) as one of the most consistent features of metastatic cells. From analysis of two independent sets of primary human tissue specimens, we determined that MSI2 expression is significantly elevated during tumor progression. Using six independent metastatic murine and human NSCLC cell line models, we demonstrated that MSI2 is essential for NSCLC invasion and metastasis in vitro and in vivo. Candidate pathway analysis and reverse-phase protein array (RPPA) screening identified EMT-associated proteins including the TGF-? type I receptor (TGFBRI), SMAD3, claudins, and the vascular endothelial growth factor receptor (VEGFR2) as strongly regulated by MSI2 in NSCLC. We directly demonstrated a functional role for several of these proteins in MSI2-induction of invasion. This proposal focuses on MSI2 regulation of VEGFR2 and its ligand, VEGF-A, given the established importance of VEGFR signaling in NSCLC, and the clinical interest of drugs targeting this pathway. We hypothesize that autocrine MSI2 regulation of VEGFR2 and VEGF-A is important for NSCLC invasion, signaling and angiogenesis, and paracrine MSI2 regulation of these proteins supports tumor angiogenesis. In this proposal, we will complement investigations of MSI2 regulation of VEGF signaling with preclinical studies addressing the importance of targeting MSI2 and VEGF signaling pathways to yield improved treatment strategies for NSCLC. In Aim 1, we will use in vitro experiments to define the autocrine and paracrine consequences of MSI2 regulation of VEGF downstream signaling. We will test if MSI2 regulates VEGFR2- dependent signaling and growth of NSCLC cells in hypoxia and whether MSI2 tumor expression affects endothelial cells growth in co-culture experiments, mechanism of MSI2 regulation of target mRNAs and evaluate relevant candidate signaling intermediates. In Aim 2, we will establish the role of MSI2-VEGF signaling in tumor growth, vascularization and invasion using both orthotopic xenograft models and in MSI2-KP transgenic mice and in human tissue microarrays. In Aim 3, we will evaluate MSI2 as a regulator of response to VEGFR2/VEGF-A inhibitors (specifically cabozantinib, and bevacizumab in combination with carboplatin) and as a drug target. In order to determine whether MSI2 is a useful drug target, we will test novel specific compounds targeting MSI2 for control of NSCLC.